Expression of Reticulomyxa filosa α- and β-tubulins in Escherichia coli yields soluble and partially correctly folded material

Linder, Stefan; Schliwa, Manfred; Kube-Granderath, Eckhard

doi:10.1016/s0378-1119(98)00142-5

Cited by 9 publications

(4 citation statements)

References 39 publications

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“…To investigate whether LdCyP or its N-terminal truncated version, like in vitro , also prevents aggregate formation under in vivo conditions, we designed vectors that simultaneously expressed both AdK and LdCyP or its truncated version in E. coli . Several workers in the past have used a similar coexpression strategy to study protein−protein interactions in vivo ( − ). However, the strategy that we adapted had several distinct advantages over others.…”

Section: Discussionmentioning

confidence: 99%

Isomerase-Independent Chaperone Function of Cyclophilin Ensures Aggregation Prevention of Adenosine Kinase Both in vitro and under in vivo Conditions

et al. 2004

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Using inactive aggregates of adenosine kinase (AdK) from Leishmania donovani as the model substrate, we recently demonstrated that a cyclophilin (LdCyP) from the same source in an isomerase-independent fashion reactivated the enzyme in vitro by disaggregating its inactive oligomers [Chakraborty et al. (2002) J. Biol. Chem. 277, 47451-47460]. Besides disrupting preformed aggregates, LdCyP also prevents reaggregation of the newly formed active protein that is generated after productive refolding from its urea-denatured state. To investigate possible physiological implications of such phenomena, a unique expression system that simultaneously induces both AdK and LdCyP in naturally AdK-deficient Escherichia coli, was developed. Both in vitro and in vivo experiments revealed that oligomerization is an inherent property of this particular enzyme. In vivo protein cross-linking studies, activity determination analysis and Ado phosphorylation experiments carried out in cells coexpressing both the proteins unequivocally demonstrated that, similar to the phenomena observed in vitro, aggregates of the enzyme formed in vivo are able to interact with both LdCyP and its N-terminal truncated form (N(22-88)DEL LdCyP) in a crowded intracellular environment, resulting in aggregation prevention and reactivation of the enzyme. Our results indicate that the isomerase-independent chaperone function of LdCyP, detected in vitro, participates in vivo as well to keep aggregation-prone proteins in a monomeric state. Furthermore, analogous to yeast/bacterial two-hybrid systems, development of this simple coexpression system may help in the confirmation of interaction of two proteins under simulated in vivo conditions.

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Section: Discussionmentioning

confidence: 99%

Isomerase-Independent Chaperone Function of Cyclophilin Ensures Aggregation Prevention of Adenosine Kinase Both in vitro and under in vivo Conditions

et al. 2004

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“…We conclude that β‐T1 aggregates rapidly in solution under the conditions used in our folding assays, as does β5 tubulin [33]. We further conclude that β‐T1 folding properties differ significantly from that of Giardia duodenalis and Reticulomyxa filosa tubulins [29,30] and are instead similar to that of higher vertebrate β‐tubulin.…”

Section: Properties Of Recombinant E Focardiiβ‐t1mentioning

confidence: 79%

“…A number of tubulin polypeptide chains from various exotic origins [29,30] have been successfully expressed in E. coli as soluble proteins. In contrast, recombinant tubulin polypeptide chains from various vertebrates [31–33] form inclusion bodies within E. coli presumably because they are misfolded.…”

Section: Properties Of Recombinant E Focardiiβ‐t1mentioning

confidence: 99%

“…However, folding was demonstrated not to proceed any further, with tubulin chains being trapped on GroEL and cpn60 [15,40]. More recently, the α‐ and β‐tubulin chains from the giant amoeba R. filosa were shown to remain soluble when expressed in E. coli following their interaction with the prokaryotic chaperonin GroEL/ES [29]; the resulting soluble tubulin is not necessarily native, however, as no assembly reaction was performed. To test whether β‐T1, which shares about 85% sequence identity with mammalian β‐tubulin, folds into a stable soluble state in the presence of GroEL or cpn60, denatured β‐T1 was incubated at 30 °C for 90 min in folding buffer containing GroEL or cpn60 in the absence or in the presence of Cof A.…”

Section: E Focardiiβ‐t1 Does Not Fold In the Presence Of The Mt‐cpn60mentioning

confidence: 99%

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Heterologous expression and folding analysis of a β‐tubulin isotype from the Antarctic ciliate Euplotes focardii

Pucciarelli

Miceli

Melki

2002

European Journal of Biochemistry

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Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1). To study the b-tubulin folding in lower eukaryotes, an isotype of b-tubulin (b-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35 S-labeled, denatured b-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric b-tubulin. We show for the first time in protozoa that b-tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardii b-T1 competes with human b5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardii b-T1 and b5 tubulin are compared. Finally, the mitochondrial chaperonin mt-cpn60 binds to b-T1 but is unable to release it in a native or quasi-native state.Keywords: protein folding; chaperonin; CCT; cpn60; protozoa.Tubulins are highly conserved proteins of eukaryotic cells involved in many essential cellular processes including intracellular transport, cell division and motility. Together with actin filaments, microtubules are the major components of the cytoskeletal lattice [1]. Microtubules are cylindrical polymers assembled from a-and b-tubulin heterodimers. In pluricellular organisms, tubulin primary sequences are encoded by a multigenic family whose products are heterogeneous proteins that can be classified as different isotypes [2]. Compelling evidence indicated that the different tubulin isotypes affect the structure and function of microtubules as well as their dynamic properties [3].Tubulin heterodimers of psychrophilic organisms are able to assemble into microtubules at temperatures below 4°C [4], while non cold-adapted microtubules disassemble. Compared with those of homeothermic animals, tubulin sequences from evolutionary distant psychrophilic organisms (as protozoa and fishes) revealed the presence of unique substitutions, some of which are common to all a-or b-tubulin chains with others variably distributed in the different isotypes. These substitutions, with particular posttranslational modifications, may be responsible of the peculiar dynamic properties of microtubules from coldliving organisms [4-6, S. Pucciarelli and C. Miceli, unpublished results].The efficient biogenesis of tubulins, as well as of actins, depends on the eukaryotic chaperonin referred to as CCT (cytosolic chaperonin containing TCP-1), TCP-1 complex, TRiC or Ct-cpn60 [7,8]. Similar to its prokaryotic counterpart GroEL, CCT has a double ring structure. However CCT has an eightfold symmetry and is composed of seven to nine distinct polypeptides (designated as a, b, c, etc.) [8] whereas GroEL has a sevenfold symmetry and is made of a single polypeptide chain. In addition, and in contrast to GroEL, CCT binds and folds a small range of misfolded polypeptides, most of which are components of the eukaryotic cytoskeleton. Misfolded actin and tubulin appear to bind to the ...

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Expression of Giardia duodenalis β-Tubulin as a Soluble Protein in Escherichia coli

MacDonald

Armson

Thompson

et al. 2001

Protein Expression and Purification

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Expression of Reticulomyxa filosa α- and β-tubulins in Escherichia coli yields soluble and partially correctly folded material

Cited by 9 publications

References 39 publications

Isomerase-Independent Chaperone Function of Cyclophilin Ensures Aggregation Prevention of Adenosine Kinase Both in vitro and under in vivo Conditions

Isomerase-Independent Chaperone Function of Cyclophilin Ensures Aggregation Prevention of Adenosine Kinase Both in vitro and under in vivo Conditions

Heterologous expression and folding analysis of a β‐tubulin isotype from the Antarctic ciliate Euplotes focardii

Expression of Giardia duodenalis β-Tubulin as a Soluble Protein in Escherichia coli

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